In this proposal, we focus no determining the regulation and biochemical properties of mammalian MAP/ERK kinase kinase 1 (MEKK1). MEKK1 was discovered as a consequence of its similarity to a yeast protein kinase in the mating pheromone response pathway. In transfected fibroblasts MEKK1 activates the c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPKs), and to a lesser extent the ERK1/2 and p38 MAP kinases. The ability to control the JNK/SAPK pathway and its biological actions in cells indicate that MEKK1 is primarily linked to responses to stresses, including inflammation and environmental insult. We isolated cDNAs containing the complete coding sequence of MEKK1, a large enzyme with the catalytic domain at the C-terminus and a 1200-residue, noncatalytic N- terminus. We have found that the N-terminal residues of MEKK1 form complexes with other signaling proteins. These interactions may lead to its upstream wiring and its downstream specificity. The behavior of MEKK1 does not fit the paradigm for phosphorylation-activated protein kinases. To understand the regulation and function of MEKK1, the research plan focuses on defining the independent domains in MEKK1, identifying additional proteins that bind to and defining binding sites for the known binding proteins, identifying regulators using molecular biological and biochemical methods and defining their mechanisms of action, and determining enzymatic and pathway specificity through mutagenesis and biochemical analysis.